Project summary Poly (ADP-ribose) polymerase ?PARP? activity plays a necessary role in DNA repair and inhibitors of PARP1 have been widely used to target cancers with impaired DNA repair machinery. In the past few years however PARP1 has also emerged as an important factor in regulating gene expression through chromatin. Targeting epigenetics is a promising approach in cancer as many drugs exist to alter epigenetic modifications. For PARP inhibitors, their application however as epigenetic drugs requires a more complete understanding of the precise function of PARP1 and its mechanism of action in regulating chromatin structure. We previously discovered that inhibition of PARP1 activity dramatically changes the expression levels of hundreds of genes, including genes involved in cancer. We found that increased levels of the Polycomb Repressive Complex 2 catalytic subunit EZH2 are responsible for some effects caused by PARP inhibition. Here we reported for the first time that (1) PARP1 and EZH2 occupancy negatively correlate across the genome; (2) PARP1 can directly modify EZH2; and (3) PARylation alters the enzymatic activity of EZH2. Based on these data we hypothesize that PARP1 and PARylation play an important and underappreciated role in EZH2 activity, and inhibitors of PARP can alter PRC2-mediated gene repression. Our findings may have significant translational implications since aberrant EZH2 activity contributes to cancer and PARP1 inhibitors are in clinical trials; however, their effects and interactions have not been explored at the functional and mechanistic levels. Here we propose to establish PARP1 and PARylation as a novel mechanism of EZH2 regulation and to determine the mechanisms and the functional relevance of PARP- mediated EZH2 inhibition. To test our hypothesis we propose : (Aim 1) To define mechanisms of PARP1 binding and PARylation in antagonizing EZH2-mediated chromatin modification we will assess EZH2 binding and H3K27me3 deposition across the genome after changes in PARP activity; (Aim 2) To establish mechanisms by which PARylation inhibits EZH2 functions we will determine the effect of PARylation on EZH2 affinity for histone and for other PRC2 proteins; (Aim 3) To evaluate the relevance of EZH2 PARylation we will characterize the effect of PAR-resistant EZH2 mutant on chromatin composition and PRC2 functions. We hypothesize that PARylation of EZH2 removes EZH2 from chromatin and that preventing PARylation results in persistent EZH2 binding and gene repression. The impact of this work extend beyond the effect of PARP1 and heterochromatin formation to provide a better insight into the global role of PARP1 in chromatin regulation. By characterizing the mechanism and the relevance of PARP1 activity on EZH2 functions we will provide a better rationale for targeting PARP1 as a treatment in cancer. In the long run, this work has the potential to improve the therapeutic application of PARP1 inhibitors for treating cancer and to identify cancer patients who can benefit by these class of drugs.